Unit: Bacteria-Cell Interactions
Director: Cossart Pascale
Our Unit investigates the molecular and cellular basis of the pathogenicity of Listeria monocytogenes, a model for the study of the intracellular parasitism. L. monocytogenes is responsible for severe food infections, for which the mortality rate is 30%. This bacterium is characterized by its ability to cross the intestinal, blood-brain or feto-placental barriers and to invade several cell types in which it multiplies. L. monocytogenes move in the cytosol of infected cells and spread from cell to cell using an original propelling process: cell actin polymerization at one pole of the bacteria. In 2004, our activity concerned the study of the bacterial and cellular components allowing bacterial invasion of cells, the identification of new virulence genes, their regulation and their anchoring mechanism at the bacterial surface, the analysis of the mechanisms allowing the crossing of the host barriers. In parallel, we investigate the actin-dependent motility of another intracellular bacterium, Rickettsia conorii, as well as its mechanism of entry into cells.
I. Entry of L. monocytogenes into epithelial cells:
Role of the internalins, InlA and InlB, two bacterial proteins involved in cell entry
InlA and its receptor, E-cadherin (D. Cabanes, M. Lecuit, S. Sousa).
We have followed the characterization of the InlA-dependent entry pathway, downstream from the interaction of InlA with E-cadherin. In addition to catenins, the role of Arp2/3 complex, Rho-GTPases, unconventional myosin VIIa and vezatin have been demonstrated (Photo 1).
InlB and its receptors gC1q-R and Met (H. Bierne, N. Khelef, M. Lecuit.)
InlB interacts with Met, the hepatocyte growth factor receptor, and gC1q-R, the C1q (complement component) receptor. We have followed the study of the signaling activated during InlB-dependent entry downstream Met. We showed that in addition to the Arp 2/3 complex, cofiline-LIM-kinase and the GTPases Rac and Cdc42, WAVE, N-WASP, Abi1 and ENA/VASP are also involved in the formation of the phagocytic cup (Photo 2). We have also showed that gC1qR is important for InlB-dependent entry, but requires Met activity. In parallel, we studied the role of InlB in vivo and demonstrated the existence of a species specificity of the InlB-Met interaction.
Other mechanisms involved in cell entry (S. Dupuis, J. Pizarro-Cerda, S. Seveau, E. Veiga)
We have also demonstrated that the two entry pathways, the InlA- and InlB-dependent pathways, require raft integrity, at the level of the E-cadherin recruitment at the entry site for InlA and at later stages for InlB. In parallel, we demonstrated the role of PI 4-Kinase in L. monocytogenes entry and that of PI4P in the recruitment of the adaptor molecule AP1 at the entry site. Finally, we have discovered that septin 9, a GTPase, which forms filaments colocalizing with filamentous actin and microtubules, is recruited by L. monocytogenes during invasion and appears to contribute to the vacuole escape. Finally, we showed that Met ubiquitination and endocytosis, induced by InlB, regulate L. monocytogenes entry into cells.
II. Identification of new virulence mechanisms for L. monocytogenes
New virulence factors have been identified by random mutagenesis or by a post-genomic approach:
Bsh (O. Dussurget), a bile salt hydrolase that favors bacterial resistance to bile and persistence in the intestinal lumen
Auto (D. Cabanes), an autolysin facilitating entry into cells, essential for several steps of infection
FbpA (D. Cabanes, S. Dramsi), a surface protein, secreted by the SecA2 secretion system, which binds fibronectin, allow bacterial adhesion to cells and regulate the production of other virulence factors, listeriolysin and InlB, most probably by acting as a chaperone
Stp (C. Archambaud, O. Dussurget), a serine/threonine phosphatase involved in virulence that targets EF-Tu and controls its activity
Two sortases (H. Bierne, C. Sabet), anchoring surface proteins via a motif LPXTG (sortase A) or NXXTX (sortase B). Inactivation of sortase A abolishes the anchoring of LPXTG proteins and attenuates virulence. Inactivation of sortase B does not affect virulence. Its exact contribution is currently studied.
III. Regulation of Listeria monocytogenes virulence gene expression
Study of the VirR/VirS regulon (P. Mandin). The two component system VirR/VirS was discovered by STM. The transcriptomal analysis of VirR/VirS showed that it control several genes, some of which are involved in the synthesis or the modification of surface components, demonstrating their essential role in infection.
Searching for new regulating non coding small RNAs (P. Mandin, F. Repoila). Several approaches are used to identify small RNAs which might be involved in virulence, among analysis of membranes of intergenic sequences and co-immunoprecipitation using antibodies specific for Hfq (a protein binding to small RNAs) that we have produced (Collaboration with J. Johansson).
IV. Epidemiology, physiopathology of infection and host responses to L. monocytogenes
Role of InlA in the crossing of host barriers (E. Huillet, M. Lecuit)
An epidemiological study showed that 96% of clinical strains and only 65% of food strains express a functional InlA (collaboration with C. Jacquet, Centre de Référence des Listeria). All strains from materno-fetal infections express a functional InlA, suggesting the implication of InlA in the crossing of the feto-placental barrier. Histological studies and analysis of the bacterial invasion of trophoblasts in culture or from tissue explants confirmed this hypothesis.
As InlA recognize the human but not the mouse E-cadherin, we are generating transgenic mice expressing the human E-cadherin instead of the mouse E-cadherin to determine the contribution of InlA in the crossing of other host barriers (Collaboration with C. Babinet, Unité de Biologie du Développement, Institut Pasteur).
Host responses to L. monocytogenes infection (O. Dussurget, M. Lecuit)
We have started a global analysis of the host response to infection by a transcriptomic approach (Collaborations with J.I. Gordon, Washington University, St Louis, MO, and P. Ricciardi-Castagnoli, University of Milano-Bicocca, Italy). We studied the intestinal response of germ-free mice expressing the human E-cadherin at the intestinal level and of epithelial and dendritic cell lines. The comparison of the gene expression patterns induced by wild type and mutants strains of L. monocytogenes is currently analyzed.
Real time analysis of murine listeriosis (O. Dussurget)
We are initiating the real time analysis of L. monocytogenes infection using a non invasive technique of imaging based on bioluminescence (Collaboration with J. Hardy and C. Contag from Stanford University, CA). This approach should allow us to perform a spatio-temporal analysis of the progression of murine listeriosis.
V. Rickettsia conorii: Another model to study actin-dependent motility and entry (E. Gouin, J. Martinez, V. Villiers)
As L. monocytogenes, Rickettsia conorii, has an intracytoplasmic motility dependent on actin polymerization. Actin filaments polymerized by R. conorii are long, unbranched, and attached to the bacteria. We have identified a gene rickA gene present in R. conorii but absent from R. prowazekii, which does not polymerize actin. The RickA protein activates directly and probably transiently the Arp2/3 complex, inducing the formation of long actin filaments. We also showed that R. conorii entry is dependent on activation of the Arp2/3 complex and that this process is regulated by Cdc42, PI 3-kinase, c-Src and cortactine.
Photo 1. Scheme of the factors and events involved in InlA-dependent entry.
Photo 2. Scheme of the factors and events involved in InlB-dependent entry.
Keywords: bacteria, virulence, cell biology, transcriptomics, mutagenesis